(1) During the current year we have continued to examine the properties of the polyglutamate metabolites of methotrexate (MTX). We have shown that, in human breast cancer cells, additional glutamyl residues are added in gamma-carboxyl linkage to MTX to form derivatives up to five peptides in length. The longer derivatives are retained by the cell, both in free form and bound to dihydrofolate reductase, when free extracellular drug is removed from the medium. The rate of disappearance of the polyglutamates is inversely related to their chain length. The longer polyglutamates progressively replace the parent compound as the major form of bound inhibitor. We have purified dihydrofolate reductase from human breast cancer cells to homogeneity and have begun to determine the binding affinity of the polyglutamates. (2) We have initiated studies of MTX-sensitive and -resistant human lung cancer cell lines, and have described the first case of unstable MTX resistance associated with double minute chromosomes and elevated DHFR levels in a tumor line from a patient treated with (and resistant to) high-dose MTX. (3) We have continued studies of melatonin physiology in women with breast cancer and have demonstrated the absence of a nocturnal peak in melatonin secretion in women with ER+ breast cancer.